Abstract: A reversed phase high performance liquid chromatography method（RP-HPLC） was developed for determination of plasma antipyrine.Plasma samples were prepared with acetonitrile.The chromatographic separation was performed on a Kromasil C18 column（250.0 mm × 4.6 mm,5 μm） at column temperature of 30 °C.The mobile phase,a mixture of 0.025 mol/L aqueous solution of sodium acetate and acetonitrile（74：26;V/V）,was delivered at a flow rate of 0.8 mL/min.The detection wavelength was 254 nm.The injection volume was 20 μL.Excellent line relationship was obtained in the range of 0.1 to 30 μg/mL（r = 0.999 5）.The average recovery rate was 93.44%-97.21%.The intra-day and inter-day RSD were less than 4.26%.